1. Field of the Invention
This invention relates to novel peptides that stimulate the production of bacteriocins by producer bacteria in the presence of inducer bacteria and to methods for using the peptides for bacteriocin production.
2. Description of the Related Art
Normal intestinal bacteria are critical to the health of any host animal. The host derives benefit through the digestive metabolic processes mediated by the native bacterial biota. From the perspective of the intestinal bacteria, competition and consequent evolution provide nutrients and living space and increase reproductive potential, enabling certain strains and species to gain survival advantage. During bacterial evolution, bacteriocin production has occurred. Bacteriocins are antagonistic to other organisms within a given competitive niche and thus provide an ecological advantage. These bacteriocins are typically low-molecular-weight polypeptides and are classified based on differences in molecular weight (Klaenhammer, FEMS Microbiol. Rev., Volume 12-39-85, 1993). These compounds can be digested easily into their component amino acids by host protease enzymes. Bacteriocins may represent a significant component of the benefits derived from competitive exclusion (Nurmi and Rantala, Nature, London, Volume 241, 210-211, 1973).
Nurmi and Rantala (1973, supra) originally described the advantages of competitive exclusion in controlling Salmonella colonization among newly hatched chicks by using an undefined bacterial flora derived from the feces of healthy adult birds. This approach is an attractive alternative to current husbandry practices involving synthetic antibiotics. A mucosal derived competitive exclusion flora was described (Stern et al., U.S. Pat. No. 5,451,400, issued September 1995) as an anaerobic culture derived from the scrapings of the intestinal mucosal linings in healthy adult hens. This undefined flora provided excellent protection against Salmonella colonization in chickens but provided only inconsistent control of Campylobacter colonization (Stern, Poult. Sci, Volume 73, 402-407, 1994). Competitive exclusion occurs within the intestinal tract of wild birds and contributes to healthy gut ecology.
Microorganisms produce a variety of compounds which demonstrate anti-bacterial properties. One group of these compounds, the bacteriocins, consists of bactericidal proteins with a mechanism of action similar to ionophore antibiotics. Bacteriocins are often active against species which are closely related to the producer of the bacteriocin. Their widespread occurrence in bacterial species isolated from complex microbial communities such as the intestinal tract, the oral, or other epithelial surfaces, suggests that bacteriocins may have a regulatory role in terms of population dynamics within bacterial ecosystems. Bacteriocins are defined as compounds produced by bacteria that have a biologically active protein moiety and bactericidal action (Tagg et al., Bacteriological Reviews, Volume 40, 722-256, 1976). Other characteristics may include: (1) narrow inhibitory spectrum of activity centered about closely related species; (2) attachment to specific cell receptors; and (3) plasmid-borne genetic determinants of bacteriocin production and of host cell bacteriocin immunity. Incompletely defined antagonistic substances have been termed “bacteriocin-like substances”. Some bacteriocins effective against Gram-positive bacteria, in contrast to Gram-negative bacteria, have wider spectrum of activity. It has been suggested that the term bacteriocin, when used to describe inhibitory agents produced by Gram-positive bacteria, should meet minimum criteria (1) being a peptide, and (2) possessing bactericidal activity (Tagg et al., supra).
In order to make commercial use of bacteriocins economically feasible, optimization of yield during production is necessary (Chen and Hoover, Comprehensive Reviews in Food Science and Food Safety, Volume 2, 82-100, 2003). Chen and Hoover state that for nisin production, it was found that in growth media, the key factors were maintenance of optimal pH and supplementation of the medium with specific nutrients for each strain or strains producing the bacteriocin.
Knutsen et al. (Journal of Bacteriology, Volume 186 (10), 3078-3085, 2004) disclose a bacteriocin inducing peptide (BIP) of 27 amino acids which induces bacteriocin production in Streptococcus pneumoniae. Diep et al. (Molecular Biology, Volume 47 (2), 483-494, 2003) discloses that a number of Gram-positive bacteria have been reported to apply a so-called peptide pheromone-based signal-transducing pathway to regulate the production of antimicrobial peptides, known as bacteriocins, from numerous lactic acid bacteria. They also disclose a peptide pheromone, PlnA, which induces bactericiocin production. Van Belkum and Stiles (Nat. Prod. Rep., Volume 17, 323-335, 2000) disclose that some bacteriocins require products of regulatory genes for their production. They state that these genes encode a secreted induction peptide and proteins that are homologous to histidine kinases and response regulators. The reference further discloses that some class II bacteriocins are induced by an induction peptide while others such as carnobacteriocin B2 and sakacin P are induced by an induction peptide or autoinduced by the synthesized bacteriocin.
While various peptides have been found which induce the production of bacteriocins in bacteria, there remains a need in the art for commercial production of bacteriocins using peptides which increase the yield of bacteriocin production in vitro. The present invention provides peptides which are different from prior art peptides and provides a method for producing large quantities of bacteriocins for commercial use.